Molecular Mechanism of Polarized Growth in Pollen Tubes

花粉管极化生长的分子机制

• 批准号: 9724047
• 负责人: Zhenbiao Yang
• 金额: $ 28.5万
• 依托单位: Ohio State University Research Foundation -DO NOT USE
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-15 至 1999-10-19
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9724047&HistoricalAwards=false
• 关键词: Molecular; Mechanism; Polarized; Growth; Pollen

9724047 Yang Tip-growing pollen tubes grow directionally toward the ovules to delivery sperms to the embryo sac and thus are critical for sexual reproduction in higher plants. Pollen tubes also present an attractive model system for the study of polarized growth, partly because they are haploid and can be easily cultured. The long-term goal of this project is to elucidate molecular pathways that govern polarized cell growth in pollen tubes. We have identified a rho-type small GTP-binding protein, Rop1, that preferentially expressed in pollen and pollen tubes and is localized to the apex of pea and Arabidopsis pollen tubes. Antibody microinjection experiments indicate that Rop1 plays a pivotal role in pollen tube growth likely by modulating Ca2+-dependent exocytosis. The goal of this proposed work is to confirm the role of Rop1 in polarized tube growth using a reverse genetics approach and to investigate underlying molecular and cellular mechanisms for the function of Rop1 in pollen tubes. The role of Rop1 will be genetically tested using transgenic Arabidopsis plants that express dominant positive and negative mutant Rop1 alleles under the control of a pollen tube-specific promoter. The transgenic pollen tubes will be analyzed for changes in tip growth and in vivo guidance. The transgenic pollen tubes expressing Rop1 dominant mutant alleles will also be analyzed for changes in the behavior of secretory vesicles using TEM and the rapid freeze and freeze substitution technique and for changes in the influx of extracellular Ca2+ and the tip-focused intracellular Ca2+-gradients using a Ca2+-selective vibrating probe and ratiometric ion imaging. These careful analyses will test the notion that Rop1 controls polarized cell growth by modulating Ca2+ signaling-dependent polarized exocytosis. To further understand the underlying mechanism for Rop1-mediated polarized growth, Rop1 effectors will be identified by cloning genes encoding Rop1 partners using interactive cloning techniques such as the yeast two-hybrid method. Rop1 effectors are expected to interact specifically with GTP-bound Rop1 (dominant positive mutant) but not with GDP-bound Rop1 (dominant negative mutant). These studies will establish a solid foundation for our long-term goal of elucidating molecular pathways that lead to directional pollen tube growth. This work may also generate a broad impact on understanding the molecular basis of polarized growth in higher organisms as well as produce useful information for manipulating sexual reproduction in higher plants, such as engineering male sterility for hybrid production. The polar growth of pollen tubes is an interesting and significant process in which a germinating pollen tube extends in a single direction to mediate fertilization and reproduction in plants. The PI has identified a protein (ROP1, GTP-binding protein) involved in the signal transduction system that appears to control the polar growth of pollen tubes through calcium fluxes. With this preliminary data as a base the PI will use mutants of ROP1 to examine the function of the protein. Using light microscopy assays the role of ROP1 in inducing a calcium fluxes will be visualized and correlated with electron microscopy of the intracellular events that are required to extend the pollen tube. ROP1 must interact with other proteins in cascade that transmits signals through the cell. Proteins that interact with ROP1 will be identified and characterized. This research is of fundamental importance in describing a critical process in the reproduction of plants. Practical applications of the research include modifying plant reproductive growth to create superior plants. ***

9724047 Yang 尖端生长的花粉管向胚珠定向生长，将精子输送到胚囊，因此对于高等植物的有性繁殖至关重要。 花粉管还为研究极化生长提供了一个有吸引力的模型系统，部分原因是它们是单倍体并且易于培养。 该项目的长期目标是阐明控制花粉管中极化细胞生长的分子途径。 我们已经鉴定出一种 rho 型小 GTP 结合蛋白 Rop1，它优先在花粉和花粉管中表达，并且位于豌豆和拟南芥花粉管的顶端。 抗体显微注射实验表明，Rop1 可能通过调节 Ca2+ 依赖性胞吐作用在花粉管生长中发挥关键作用。 这项工作的目标是使用反向遗传学方法确认 Rop1 在极化管生长中的作用，并研究 Rop1 在花粉管中功能的潜在分子和细胞机制。 Rop1 的作用将使用转基因拟南芥植物进行基因测试，这些植物在花粉管特异性启动子的控制下表达显性阳性和阴性突变体 Rop1 等位基因。 将分析转基因花粉管的尖端生长和体内引导的变化。 表达 Rop1 显性突变等位基因的转基因花粉管还将使用 TEM 和快速冷冻和冷冻替代技术分析分泌囊泡行为的变化，并使用Ca2+选择性振动探针和比例离子成像。 这些仔细的分析将检验 Rop1 通过调节 Ca2+ 信号依赖性极化胞吐作用来控制极化细胞生长的概念。 为了进一步了解 Rop1 介导的极化生长的潜在机制，将使用交互式克隆技术（例如酵母双杂交方法）克隆编码 Rop1 伴侣的基因来鉴定 Rop1 效应子。 Rop1 效应子预计会与 GTP 结合的 Rop1（显性正突变体）特异性相互作用，但不会与 GDP 结合的 Rop1（显性负突变体）相互作用。 这些研究将为我们阐明导致花粉管定向生长的分子途径的长期目标奠定坚实的基础。 这项工作还可能对理解高等生物极化生长的分子基础产生广泛的影响，并为操纵高等植物的有性生殖（例如为杂交生产工程雄性不育）提供有用的信息。 花粉管的极性生长是一个有趣且重要的过程，其中发芽的花粉管沿单一方向延伸以介导植物的受精和繁殖。 PI 鉴定出一种参与信号转导系统的蛋白质（ROP1，GTP 结合蛋白），该蛋白质似乎通过钙流控制花粉管的极性生长。以此初步数据为基础，PI 将使用 ROP1 突变体来检查该蛋白质的功能。使用光学显微镜检测，ROP1 在诱导钙通量中的作用将被可视化，并与延长花粉管所需的细胞内事件的电子显微镜相关。 ROP1 必须与其他蛋白质级联相互作用，通过细胞传递信号。与 ROP1 相互作用的蛋白质将被鉴定和表征。这项研究对于描述植物繁殖的关键过程至关重要。该研究的实际应用包括改变植物的生殖生长以创造优质植物。 ***